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Team:HokkaidoU Japan/Notebook/Pre experiment/Stem replacement
From 2014.igem.org
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| - | R0010 on ice 30min each of 5.0µL DNA to DH5α 42& | + | R0010 on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min |
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</div> | </div> | ||
Revision as of 12:50, 17 October 2014
hogehoge
Stem Region Replacement
-
Start
-
PCR
pHN1257 stem-f Tm:70.6 terminator-r Tm:71.4 KOD FX NEO -
Transformation
R0010 on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min -
Digestion
Cut R0010 with PstI, SpeI (using 10xH buffer) and pHN1257 with XbaI, PstI (using 10xM buffer) -
Ligation
Ligate R0010 with pHN1257 -
Transformation
Ligation products on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min -
Colony PCR
R0010-stem-dT 100up-f pHN1257terminator-r -
mini-Prep
R0010-stem-dT -
PCR(confirm)
R0010-stem-dT 100up-f pHN1257terminator-r KOD FX NEO -
Complete!
